Effects Of Nrf2 On The Expression Of NOX1 Gene In TNF-α-induced Type Ⅱ Alveolar Epithelial Cells

BackgroundAcute lung injury and acute respiratory distress syndrome are high morbidity and mortality lung diseases,because of the lack of effective treatment.Direct or indirect injury to the lung parenchyma results in damage of alveoli type II epithelial cells and endothelial cells of the pulmonary capillaries,leading to loss of the alveolar-capillary memebrane barrier.Without this barrier,fluid leaks into the alveoli,causing the pulmonary edema and respiratory failure.Uncontrolled inflammatory reaction and excessive oxidative stress are one of the important mechanisms that cause alveolar epithelial cell injury in ALI/ ARDS patients.Thus,except for anti-inflammatory treatment,maintaining oxidative stress as well as inflammatory response at an appropriate level should benefit to the protection of ALI.A variety of inflammatory factors causes an inflammatory cascade response,especially TNF-α,which play an important role on acute lung injury.The previous research show that,the level of IL-1 and IL-6 were significantly up-regulated in A549 stimulated with TNF-α,and the concentration of reactive oxygen species was increased as well.At the same time,the activation of NF-κB pathway leads to the up-regulation of the rate of apoptosis in A549.The genertation of ROS and consumption of the antioxidants break the balance of oxidative stress.The NOX is known as an important source of ROS.The function and expression of NOX isoforms have tissue and cell specificity,which affect the generation of ROS.NOX1 is expressed in alveolar epithel cells,and make contribution to ALI.Reducing the level of ROS drived from NOX can be an effective way to lighten the damage of oxidative stress.Nuclear factor-E2 related factor 2（Nrf2）,a key transcription factor that contributes to the expression of antioxidant genes.Nrf2 binds to Keap1,and translocates into the nucleus to activate the transcription of its target genes,including oxidative stress,inflammation,apoptosis,autophagy,and so on.In addition,numerous researches have demonstrated the significance of Nrf2 in the protection against ALI,as the activation of Nrf2 prevents the occurrence or mitigates the severity of ALI/ARDS.However,the effect of Nrf2 on the NOX is not clear.There is a specific Nrf2 binding site predicted by Alibaba 2.1 on NOX1 promoter.The expression of Nrf2,as well as NOX1 were increased in A549 induced by TNF-α in our early study.Therefore,it is necessary for us to study wheather the Nrf2 is involved in the regulations of NOX1/ROS,which may in favor of the research on the ALI/ARDS therapy.Objectives1.To investigate the effects of Nrf2 on the damege of inflammatory and oxidative stress in A549 induced by TNF-α.2.To study whether the Nrf2 was involved in the regulation of NOX1.Methods1.The effects of TNF-α on NOX1 and Nrf2 gene expression in alveolar type II epithelial cellsA549 were cultured in RPMI-1640 supplemented with 10% FBS,then they were treated with different concentration of TNF-α(0/2.5/5/10/20/40 ng.ml^-1)for different times（0/6/12/24/36/48h）.Then the levels of NOX1,Nrf2 m RNA and protein were measured by real time PCR and Western Blotting respectively.2.The Protection of Nrf2 on the damage of inflammatory and oxidative stress in alveolar type II epithelial cells induced by TNF-αSix experimental groups were as fol Iows: A.control group,B.TNF-α group,C.negative si RNA group,D.Nrf2 si RNA group,E.empty vector group,F.Nrf2 high-expression vector group.The levels of IL-6 and IL-8 were measured by ELISA,the expression of Nrf2 and NF-κB were detected by real time PCR and Western Blotting,the level of ROS was detected by DCFH-DA,the concentration of MDA and TAOC were detected by kits using colorimetry.3.Nrf2 regulates the expression of NOX1 in A549Nrf2 binding site in the proximal promoter（1500 bp）of NOX1 gene was predicted by Alibaba 2.1.The whole NOX1 promoter and NOX1 promoter with deletion fragment were inserted to the p GL3 basic vector to construct recombinant vectors respectively,named as “ p GL3-NOX1-1500” and “ p GL3-NOX1-1489”.After transfection with the vetors,the A549 cells were irritated with TNF-α and the level of luciferase was detected by the instrument.4.Statistical analysisStatistical analyses were performed using SPSS21.0.The data were represented as “ X ±S ”,and analyzed by independent-samples t-test and one-way ANOVA.P<0.05 was considered to be statistically significant.Results1.The effects of TNF-α on NOX1 and Nrf2 gene expression in alveolar type II epithelial cellsThe level of NOX1,Nrf2 mRNA and protein were increased after stimulated with 10μg/L TNF-α for 24 h,compared with control group,P <0.05.2.The Protection of Nrf2 on the damage of inflammatory and oxidative stress in alveolar type II epithelial cells induced by TNF-α2.1 The level of Nrf2 m RNA and protein were increased in TNF-α group,compared with control group,P<0.05.The expression of Nrf2 m RNA and protein in Nrf2 si RNA group were decreased,P<0.05,While Nrf2 high-expression vector group were increased,P <0.05.2.2 The concentrations of IL-6 and IL-8 in the culture supernatant were higher in TNF-α group,compared with control group,P <0.05.And,it was higher in Nrf2 si RNA group,P <0.05,while lower in Nrf2 high-expression vector group,P <0.05.2.3 The level of MDA and ROS were increased in TNF-α group,compared with control group,P <0.05.And,it was higher in Nrf2 si RNA group,P <0.05,while lower in Nrf2 high-expression vector group,P <0.05.2.4 The concentrations of TAOC was increased in TNF-α group,compared with control group,P <0.05.It was lower in Nrf2 si RNA group,but higher in Nrf2 high-expression vector group,P <0.05.2.5 The expression of NF-κB m RNA and protein were increased in TNF-α group,compared with control group,P <0.05.NF-κB m RNA and protein in Nrf2 si RNA group were upregulated,P <0.05.While Nrf2 high-expression vector group were decreased,P <0.05.3.Nrf2 regulates the expression of NOX1 in A5493.1 Compared with control group,the expression of NOX1 m RNA and protein were higher in TNF-α group,P <0.05.The expression in Nrf2 si RNA group were higer than that in TNF-α group and negative si RNA group,P <0.05.However,Nrf2 high-expression vector group were lower than that in TNF-α group and empty vector group,P <0.05.3.2 One potential Nrf2 element on NOX1 promoter was predicted by Alibaba 2.1.3.3 The sequence of the recombinant vectors “ p GL3-NOX1-1500” and “ p GL3-NOX1-1489” were confirmed to be corret.3.4 The level of luciferase in “ p GL3-NOX1-1489”group was higher than that in control group,P <0.05,while lower than that in “p GL3-NOX1-1500” group,P <0.05.ConclusionTNF-α could increase the expression of NOX1 and Nrf2 in alveolar type II epithelial cells effectively.Nrf2 could reducing the damege of inflammation and oxidative stress in A549 cells stimulated by TNF-α.In addition,Nrf2 may regulate the transcription of NOX1 gene by binding to the NOX1 promoter specfic binding site,then affects the generation of ROS.